Our research focuses on events in the exocytotic pathway that occur in the highly specialized domain of the plasma membrane-cytoplasm interface. This region is superbly imaged by total internal reflection fluorescence microscopy (TIRFM), a core technique that we use extensively in our studies. This proposal is based upon the hypothesis that the mobility characteristics of granule lumenal contents and of granule membrane proteins shape the secretory response. The proposal will provide fundamental new insights concerning secretory granule structure and function in exocytosis and will provide the first quantitative measures of the rotational and translational mobility of granule lumenal and membrane proteins of individual granules. The rotational and translational mobility of lumenal and granule membrane proteins and of individual granules will be measured by novel combinations of TIRFM, polarization and FRAP. There are several related goals in the proposal: 1) to understand the physical state of the granule lumen and reveal the role of lumenal viscosity in determining the rates of protein and catecholamine release, 2) to determine the translational mobility of granule membrane proteins and whether the mobility permits recruitment to the fusion site on the granule membrane, 3) to determine the rotational mobility of individual granules in order to better define the tethered and/or caged state of the granules before fusion, and 4) and to determine whether the increase in granule travel immediately before fusion reflects a combination of translational and rotational motion that permits the granule to 'roll'into a fusion competent interaction with the plasma membrane. PUBLIC HEALTH RELEVANCE: Our research focuses on the mechanisms by which hormones and neurotransmitters are secreted from cells including neurons. These processes underlie the function of the cardiovascular, endocrine and nervous systems. The process (exocytosis) occurs by the stimulated fusion of a storage vesicle or granule with the cell (plasma) membrane. This proposal will establish new, powerful optical techniques to probe the structure and function of secretory granules in living and secreting cells. The findings will help us understand the mechanism of granule membrane fusion with the cell membrane and the factors which control release of granule contents. These studies have direct application to processes that are required for health and malfunction in disease.